U.S. patent application number 14/771358 was filed with the patent office on 2016-03-03 for culture system for pluripotent stem cells and method for subculturing pluripotent stem cells.
The applicant listed for this patent is Kyoto University, Nipro Corporation. Invention is credited to Norio Nakatsuji, Masakatsu Takeuchi, Daiki Tateyama, Yoshihiro Yoshikawa.
Application Number | 20160060588 14/771358 |
Document ID | / |
Family ID | 51491103 |
Filed Date | 2016-03-03 |
United States Patent
Application |
20160060588 |
Kind Code |
A1 |
Nakatsuji; Norio ; et
al. |
March 3, 2016 |
CULTURE SYSTEM FOR PLURIPOTENT STEM CELLS AND METHOD FOR
SUBCULTURING PLURIPOTENT STEM CELLS
Abstract
A culture system has a culture bag for suspending and culturing
pluripotent stem cells in a culture medium; a waste liquid
container for storing used culture medium that is connected to the
culture bag; a fresh medium container for storing a fresh culture
medium that is connected to the culture bag; three-way stopcocks
for switching flow channels from the culture bag to the waste
liquid container or the fresh culture medium container, etc.; a
trap portion for trapping the pluripotent stem cells in the culture
medium in the flow toward the waste liquid container side; and a
filter portion for subculturing that is formed in fourth and fifth
flow channels in parallel to a first flow channel. The first flow
channel connects the trap part to the culture bag, and has a mesh
by which a cell mass of the pluripotent stem cells can be divided.
A pump is used to pump the liquid in the individual flow
channels.
Inventors: |
Nakatsuji; Norio; (Kyoto,
JP) ; Yoshikawa; Yoshihiro; (Osaka, JP) ;
Takeuchi; Masakatsu; (Osaka, JP) ; Tateyama;
Daiki; (Osaka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kyoto University
Nipro Corporation |
Kyoto
Osaka |
|
JP
JP |
|
|
Family ID: |
51491103 |
Appl. No.: |
14/771358 |
Filed: |
February 20, 2015 |
PCT Filed: |
February 20, 2015 |
PCT NO: |
PCT/JP2014/054066 |
371 Date: |
November 5, 2015 |
Current U.S.
Class: |
435/366 ;
435/297.2; 435/383 |
Current CPC
Class: |
C12M 25/02 20130101;
C12M 29/00 20130101; C12M 29/04 20130101; C12M 23/14 20130101; C12M
41/00 20130101; C12M 23/58 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12M 1/12 20060101 C12M001/12; C12M 1/34 20060101
C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 6, 2013 |
JP |
2013-044727 |
Claims
1. A culture system for pluripotent stem cells comprising: a
culture container for suspending and culturing pluripotent stem
cells in a culture medium; a waste liquid container which is
connected to the culture container by flow channels through which
liquid can flow and which stores a used culture medium flowing out
of the culture container; a fresh culture medium container which is
connected to the culture container by flow channels through which
liquid can flow and which stores a fresh culture medium to be
supplied to the culture container; first switching portions which
switch the flow channels from the culture container to the waste
liquid container or the fresh culture medium container; a trap
portion which is provided in the flow channel on a side of the
culture container with respect to the first switching portions and
which traps the pluripotent stem cells in the culture medium in
flow to a side of the waste liquid container; a subculturing filter
portion which is provided in flow channels provided in parallel to
a flow channel connecting the trap portion and the culture
container between the trap portion and the culture container and
which has a mesh capable of dividing a cell mass of the pluripotent
stem cells; and second switching portions which switch flow
channels from the trap portion to the culture container or the
subculturing filter portion.
2. The culture system for pluripotent stem cells according to claim
1, further comprising: a backwashing container which is provided in
a flow channel connecting the culture container and the
subculturing filter portion in such a manner as to allow passage of
liquid and which stores a backwashing culture medium to be supplied
to the subculturing filter portion.
3. The culture system for pluripotent stem cells according to claim
1, further comprising: a control portion for controlling a flow
rate of liquid in each of the flow channels, wherein the control
portion sets a flow rate per unit square cm of liquid flowing
through the mesh of the subculturing filter portion to 95 mL or
more per minute.
4. The culture system for pluripotent stem cells according to claim
1, further comprising: a pump for letting liquid flow through each
of the flow channels; and a control portion which controls drive of
the pump, wherein the control portion drives the pump at a fixed
flow rate of 90 mL or more per minute at least in flow from the
trap portion to the subculturing filter portion.
5. The culture system for pluripotent stem cells according to claim
1, wherein the pluripotent stem cells are ES cells or iPS
cells.
6. The culture system for pluripotent stem cells according to claim
1, the pluripotent stem cells are human-derived pluripotent stem
cells.
7. A method for subculturing pluripotent stem cells comprising: a
culturing step of culturing suspending and culturing a cell mass of
pluripotent stem cells; a first dividing step of letting the cell
mass of the cultured pluripotent stem cells flow into a mesh, which
is provided in a flow channel through which a culture medium can
flow, in one direction together with a culture medium to thereby
divide the cell mass, and then accommodating the divided cell
masses in a culture container; a backwashing step of closing a flow
channel to the culture container, and then letting the culture
medium flow into the mesh in an opposite direction to the direction
in the dividing process to remove a cell mass clogging the mesh;
and a second dividing step of opening the flow channel to the
culture container, letting the removed cell mass flow in one
direction together with the culture medium to thereby divide the
cell mass, and then accommodating the divided cell masses in the
culture container.
8. The method for subculturing pluripotent stem cells according to
claim 7, wherein the pluripotent stem cells are ES cells or iPS
cells.
9. The method for subculturing pluripotent stem cells according to
claim 7, wherein the pluripotent stem cells are human-derived
pluripotent stem cells.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a system for culturing
pluripotent stem cells and a method for subculturing the
pluripotent stem cells.
BACKGROUND
[0002] The pluripotent stem cells capable of infinitely
proliferating without causing cancerization and the like and having
pluripotency have been expected to be applied to cell
transplantation treatment, drug discovery screening, and the
like.
[0003] Heretofore, a human pluripotent stem cell line has been
proliferated and maintained by plate culture of causing the human
pluripotent stem cell line to adhere to feeder cells, various
polymers, or the like. However, a technique of stably culturing a
large amount of high quality human pluripotent stem cells has not
been established yet. In particular, a former method including
causing the cell line to adhere to a culture container, and then
proliferating the same by subculturing has been insufficient for
preparing a large amount of pluripotent stem cells which are
necessary for practical use. For example, the optimal adhesion
substrate material for human pluripotent stem cells in terms of
quality and cost has not been developed. Moreover, complicated many
stages have been necessary for the subculturing, and thus
disadvantageous steps in terms of safety and cost, such as enzyme
treatment, have been included.
[0004] Recently, a suspension culture method which does not require
an adhesion substrate has been reported (Patent Literatures 1 to
5). According to the suspension culture method, three-dimensional
culture can be performed, and therefore a large amount of
pluripotent stem cells can be cultured in a smaller space.
[0005] As a method for substituting the enzyme treatment in
subculturing, a method including causing a cell mass of pluripotent
stem cells to pass through a micro grid for dividing the cell mass
has been devised (Patent Literature 6).
CITATION LIST
Patent Literatures
[0006] Patent Literature 1: International publication No. WO
2011/058558
[0007] Patent Literature 2: International publication No. WO
2009/116951
[0008] Patent Literature 3: International publication No. WO
2008/120218
[0009] Patent Literature 4: International publication No. WO
2008/015682
[0010] Patent Literature 5: International publication No. WO
2007/002086
[0011] Patent Literature 6: Japanese Unexamined Patent Application
Publication (Translation of PCT Application) No. 2010-526530
SUMMARY OF INVENTION
Technical Problem
[0012] However, a system capable of automating the culture of a
large amount of pluripotent stem cells which are necessary for
practical use has not been developed yet. Moreover, when the method
including causing a cell mass of pluripotent stem cells to pass
through a micro grid for dividing has been adopted for culturing a
large amount of pluripotent stem cells, a problem that the micro
grid has been clogged, so that appropriate and efficient
subculturing has not been realized has occurred.
[0013] The present invention has been made in view of the problems
described above. It is an object of the present invention to
provide a system suitable for culturing a large amount of
pluripotent stem cells.
[0014] It is another object of the present invention to provide a
method suitable for subculturing a large amount of pluripotent stem
cells.
Solution to Problem
[0015] (1) A culture system for pluripotent stem cells according to
the present invention has a culture container for suspending and
culturing pluripotent stem cells in a culture medium, a waste
liquid container which is connected to the culture container by
flow channels through which liquid can flow and which stores a used
culture medium flowing out of the culture container, a fresh
culture medium container which is connected to the culture
container by flow channels through which liquid can flow and which
stores a fresh culture medium to be supplied to the culture
container, first switching portions which switch the flow channels
from the culture container to the waste liquid container or the
fresh culture medium container, a trap portion which is provided in
the flow channel on the side of the culture container with respect
to the first switching portions and which traps the pluripotent
stem cells in the culture medium in the flow to the side of the
waste liquid container, a subculturing filter portion which is
provided in flow channels provided in parallel to a flow channel
connecting the trap portion and the culture container between the
trap portion and the culture container and which has a mesh capable
of dividing a cell mass of the pluripotent stem cells, and second
switching portions which switch flow channels from the trap portion
to the culture container or the subculturing filter portion.
[0016] When exchanging culture media in culturing pluripotent stem
cells, a culture medium is made to flow out of the culture
container into the waste liquid container. Although the pluripotent
stem cells flow out of the culture container together with the
culture medium, the pluripotent stem cells are trapped by the trap
portion, and therefore the pluripotent stem cells do not flow into
the waste liquid container. After the first switching portion is
switched, a fresh culture medium flows out of the fresh culture
medium container into the culture container. Due to the fact that
the fresh culture medium passes through the trap portion toward the
culture container, the pluripotent stem cells trapped by the trap
portion flow into the culture container together with the fresh
culture medium. In this operation, the second switching portion is
switched to the flow channel to the culture container not via the
subculturing filter portion from the trap portion. Thus, the
culture medium of the culture container is exchanged, and then the
pluripotent stem cells are cultured in the fresh culture
medium.
[0017] In subculturing, a culture medium is made to flow out of the
culture container into the waste liquid container, and then the
pluripotent stem cells are trapped by the trap portion similarly as
described above. Before a fresh culture medium is made to flow out
of the fresh culture medium container into the culture container,
the second switching portion is switched to the flow channel to the
culture container through the subculturing filter portion from the
trap portion. The cell mass of the pluripotent stem cells which
reaches the subculturing filter portion from the trap portion
together with the fresh culture medium is divided when passing
through the mesh. The cell masses of the pluripotent stem cells
which are divided into a size suitable for subculturing flows into
the culture container together with the fresh culture medium. Thus,
the pluripotent stem cells are subcultured.
[0018] (2) The culture system for pluripotent stem cells according
to the present invention may further have a backwashing container
which is provided in a flow channel connecting the culture
container and the subculturing filter portion in such a manner as
to allow passage of liquid and which stores a backwashing culture
medium to be supplied to the subculturing filter portion.
[0019] In subculturing, the cell mass of the pluripotent stem cells
which reaches the subculturing filter portion from the trap portion
together with the fresh culture medium is divided when passing
through the mesh but there is a cell mass which clogs the mesh
without being divided. Due to the fact that the backwashing culture
medium is made to flow into the mesh from the backwashing
container, the clogging cell masses are made to flow to the
backwashing culture medium to be separated from the mesh. Thus, the
clogging of the mesh can be eliminated.
[0020] (3) The culture system for pluripotent stem cells according
to the present invention may further have a control portion for
controlling the flow rate of liquid in each of the flow channels,
in which the control portion may set the flow rate per unit square
cm of liquid passing through the mesh of the subculturing filter
portion to 95 mL or more per minute.
[0021] In subculturing, by setting the flow rate per unit square cm
of the mesh of the subculturing filter portion to 95 mL or more per
minute, the recovery rate of the pluripotent stem cells
improves.
[0022] (4) The culture system for pluripotent stem cells according
to the present invention may further have a pump for letting liquid
flow through each of the flow channels, and a control portion which
controls the drive of the pump, in which the control portion may
drive the pump at a fixed flow rate of 90 mL or more per minute at
least in the flow from the trap portion to the subculturing filter
portion.
[0023] In subculturing, by letting the culture medium flow into the
subculturing filter portion at a fixed flow rate of 90 mL or more
per minute, the recovery rate of the pluripotent stem cells
improves.
[0024] (5) As the pluripotent stem cells, ES cells or iPS cells are
mentioned.
[0025] (6) As the pluripotent stem cells, human-derived pluripotent
stem cells are mentioned.
[0026] (7) A method for subculturing pluripotent stem cells
according to the present invention includes a culturing process of
suspending and culturing a cell mass of pluripotent stem cells, a
first dividing process of letting the cell mass of the cultured
pluripotent stem cells flow into a mesh, which is provided in a
flow channel through which a culture medium can flow, in one
direction together with a culture medium to thereby divide the cell
mass, and then accommodating the divided cell masses in a culture
container, a backwashing process of closing a flow channel to the
culture container, and then letting the culture medium flow into
the mesh in an opposite direction to the direction in the dividing
process to remove a cell mass clogging the mesh, and a second
dividing process of opening the flow channel to the culture
container, letting the removed cell mass flow in one direction
together with the culture medium to thereby divide the cell mass,
and then accommodating the divided cell masses in the culture
container.
[0027] Thus, the cell mass clogging the mesh in the first dividing
process is separated from the mesh in the backwashing process to
eliminate the clogging, and the cell mass separated from the mesh
in the second dividing process can be divided, and therefore the
recovery rate of the pluripotent stem cells in the subculturing
improves.
[0028] (8) As the pluripotent stem cells, ES cells or iPS cells are
mentioned.
[0029] (9) As the pluripotent stem cells, human-derived pluripotent
stem cells are mentioned.
Advantageous Effects of Invention
[0030] According to the culture system for pluripotent stem cells
according to the present invention, a large amount of pluripotent
stem cells can be cultured and an automated system can be
realized.
[0031] Moreover, according to the method for subculturing
pluripotent stem cells of the present invention, clogging of a mesh
is suppressed and subculturing of a large amount of pluripotent
stem cells can be realized.
BRIEF DESCRIPTION OF DRAWINGS
[0032] FIG. 1 is a schematic view illustrating the outline of a
culture system 10 according to an embodiment of the present
invention.
[0033] FIG. 2 is a cross sectional view illustrating the internal
structure of a trap portion 14.
[0034] FIG. 3 is a cross sectional view illustrating the internal
structure of a subculturing filter portion 15.
[0035] FIG. 4 shows the recovery rate of cells to the flow rate of
a culture medium passing through a mesh 31.
[0036] FIG. 5 show the distributions of the sizes of cell masses
passing through the mesh 31.
[0037] FIG. 6 shows the recovery rate of cells to the opening ratio
of the mesh 31.
EMBODIMENT FOR CARRYING OUT THE INVENTION
[0038] Hereinafter, an embodiment of the present invention is
described with reference to the drawings. This embodiment is merely
an example of the present invention, and it is a matter of course
that this embodiment may be altered as appropriate in the range
where the scope of the present invention is not altered.
[Culture System 10]
[0039] As illustrated in FIG. 1, in a culture system 10 for
pluripotent stem cells, a culture bag 11, a waste liquid container
12, a fresh culture medium container 13, a trap portion 14, a
subculturing filter portion 15, a pump 16, and backwashing
containers 17 and 18 are connected by flow channels.
[0040] The culture bag 11 is a bag formed with resin capable of
suspending and culturing pluripotent stem cells in a culture medium
and capable of sealing liquid in the internal space. A resin sheet
configuring the outer wall of the culture bag 11 is preferably one
having high permeability of gas, such as carbon dioxide. The
culture bag 11 is provided with a port, which allows a fluid, such
as a culture medium, to flow into/out of the internal space, as
appropriate. The culture bag 11 has a rectangular outer shape as
viewed in plane but the shape is not particularly limited. The
shape and the capacity are set in consideration of the capacity of
a culture medium to be charged, operability, and the like.
[0041] Although not illustrated in FIG. 1, the culture bag 11 is
accommodated in an incubator or the like in such a manner that the
environment around the culture bag 11 has a temperature and
humidity suitable for culturing. In order to prevent aggregation of
cell masses of pluripotent stem cells, a mechanism of changing the
attitude of the culture bag 11 may be provided as necessary.
[0042] The waste liquid container 12 is a container capable of
storing a used culture medium and the like which are made to flow
out of the culture bag 11 for exchanging culture media. The shape
and the capacity of the waste liquid container 12 are set as
appropriate according to the capacity of the culture bag 11, the
days while the used culture medium is to be stored, and the
like.
[0043] The fresh culture medium container 13 is a container capable
of storing a fresh culture medium to be supplied to the culture bag
11 in exchanging culture media. The shape and the capacity of the
fresh culture medium container 13 are set as appropriate according
to the capacity of the culture bag 11, the frequencies of
exchanging culture media, and the like.
[0044] As illustrated in FIG. 2, the trap portion 14 traps the cell
mass of the pluripotent stem cells flowing into the trap portion 14
together with the culture medium, and then lets only the culture
medium flow out of the trap portion 14. In the trap portion 14, an
inflow port 21 is formed in the lower side of an almost
tubular-shaped body 20 and an outlet port 22 is formed in the upper
side of the body 20. The inflow port 21 leads to the internal space
of the body 20. The cell mass of pluripotent stem cells can flow
into the internal space of the body 20 together with the culture
medium through the inflow port 21.
[0045] The outlet port 22 leads to the internal space of the body
20, and a mesh 23 is provided in the outlet to the internal space
of the body 20. The mesh 23 has a thin film shape in which a large
number of pores having a size which does not allow passage of the
cell mass of the pluripotent stem cells are formed. The cell mass
of the pluripotent stem cells flowing into the internal space of
the body 20 together with the culture medium from the inflow port
21 stays in the lower side of the internal space of the body 20 by
own weight. The cell mass of the pluripotent stem cells which
reaches the upper side in the internal space of the body 20 is
obstructed by the mesh 23, and does not flow out of the outlet port
22.
[0046] Although not illustrated in each figure, in order to prevent
aggregation of the cell masses of the pluripotent stem cells and
clogging of the mesh 23, the trap portion 14 may be provided with a
mechanism of elastically deforming the body 20 or applying
vibration to the mesh 23 as necessary.
[0047] As illustrated in FIG. 3, the subculturing filter portion 15
divides the cell mass of the pluripotent stem cells flowing into
the subculturing filter portion 15 together with the culture
medium. The subculturing filter portion 15 has a body 30
accommodating a mesh 31. The body 30 has an inflow port 32 and an
outlet port 33. The inflow port 32 and the outlet port 33 lead to
the internal space of the body 30. The mesh 31 is provided in the
internal space of the body 30 in such a manner as to separate a
flow channel leading to the outlet port 33 from the inflow port
32.
[0048] Materials of the mesh 31 are not particularly limited
insofar as the materials can be sterilized, and, for example,
synthetic resin, such as nylon and polyethylene terephthalate, and
metals, such as stainless steel, may be selected. The mesh 31
formed with stainless steel is preferable because the thickness of
fibers configuring the mesh 31 can be reduced and the opening ratio
(proportion of openings (pores) per unit area of the mesh 31) can
be increased. When the opening ratio of the mesh 31 is larger, the
recovery rate of the pluripotent stem cells to be collected by the
culture bag 11 tends to be better.
[0049] The pore size of the mesh 31 may be set in such a manner
that the average diameter of the cell masses after division is
about 80 to 120 .mu.m and preferably about 80 .mu.m. For example,
in the case of a stainless steel mesh, the pore size is about 40 to
100 .mu.m, preferably 50 to 70 .mu.m, and more preferably about 50
.mu.m. The term "about" used herein means that .+-.10% is
permitted. The shape and the like of the mesh 31 are not
particularly limited. It is preferable for the mesh 31 to have a
thickness and a shape which do not damage cells as much as
possible. When the area of a region through which the cell mass can
pass in the mesh 31 is larger, the number of the cell masses
increases but clogging is hard to occur, and thus it is
preferable.
[0050] The pump 16 lets a culture medium and the like flow in each
flow channel and a peristaltic pump is mentioned as a typical
example. Although not illustrated in each figure, the pump 16 has
an arithmetic unit as a control portion, which operates in such a
manner that the pump 16 lets a culture medium and the like flow at
a flow rate according to the input flow rate.
[0051] In this embodiment, the pump 16 is used as a drive source of
letting a culture medium and the like flow in each flow channel.
However, in place of the pump 16, for example, a culture medium may
be made to flow out of the culture bag 11 or a culture medium may
be made to flow into the waste liquid container 12 and the like by
compressing and deforming the culture bag 11 and the like or
fluctuating the pressure of the air layer of the waste liquid
container 12 and the like. Moreover, by changing the relative
height relationship between the culture bag 11, and the waste
liquid container 12 and the fresh culture medium container 13 by
utilizing the gravity, a culture medium and the like may be made to
flow between the culture bag 11, and the waste liquid container 12
and the fresh culture medium container 13, for example.
[0052] Hereinafter, flow channels connecting each of the culture
bag 11, the waste liquid container 12, the fresh culture medium
container 13, the trap portion 14, the subculturing filter portion
15, the pump 16, and the backwashing containers 17 and 18 are
described. Each flow channel is configured from a resin tube, for
example, and the internal diameter, the length, and the like are
selected as appropriate according to the flow amount, the flow
rate, and the like. The tubes configuring the flow channels may be
configured from one tube or may be configured by connecting a
plurality of tubes with a joint or the like.
[0053] The culture bag 11 is connected to the trap portion 14 by a
first flow channel 41. The first flow channel 41 connects a port of
the culture bag 11 and the inflow port 21 of the trap portion 14.
The trap portion 14 is connected to the waste liquid container 12
by a second flow channel 42. The second flow channel 42 connects
the outlet port 22 of the trap portion 14 and the waste liquid
container 12. The pump 16 is provided in the second flow channel
42. The pump 16 sends liquid in one direction in the second flow
channel 42 by selectively pulsating the tube configuring a part of
the second flow channel 42 to the trap portion 14 side or the waste
liquid container 12 side.
[0054] A three-way stopcock 51 is provided between the waste liquid
container 12 and the pump 16 in the second flow channel 42. The
three-way stopcock 51 is connected to the fresh culture medium
container 13 by a third flow channel 43. By switching the three-way
stopcock 51, the trap portion 14 is selectively connected to the
waste liquid container 12 or the fresh culture medium container
13.
[0055] Two three-way stopcocks 52 and 53 are provided in the first
flow channel 41. The three-way stopcock 52 is connected to the
subculturing filter portion 15 by a fourth flow channel 44. The
fourth flow channel 44 connects the three-way stopcock 52 and the
inflow port 32 of the subculturing filter portion 15. By switching
the three-way stopcock 52, the trap portion 14 is selectively
connected to the culture bag 11 or the subculturing filter portion
15.
[0056] The three-way stopcock 53 is connected to the subculturing
filter portion 15 by a fifth flow channel 45. The fifth flow
channel 45 connects the three-way stopcock 53 and the outlet port
33 of the subculturing filter portion 15. By switching the
three-way stopcock 53, the culture bag 11 is selectively connected
to the trap portion 14 or the subculturing filter portion 15.
[0057] A three-way stopcock 54 is provided between the trap portion
14 and the pump 16 in the second flow channel 42. The three-way
stopcock 54 is connected to the backwashing container 17 by a sixth
flow channel 46. By switching the three-way stopcock 54, the pump
16 is selectively connected to the trap portion 14 or the
backwashing container 17.
[0058] A three-way stopcock 55 is provided between the culture bag
11 and the subculturing filter portion 15 in the fifth flow channel
45. The three-way stopcock 55 is connected to the backwashing
container 18 by a seventh flow channel 47. By switching the
three-way stopcock 55, the subculturing filter portion 15 is
selectively connected to the culture bag 11 or the backwashing
container 18.
[0059] Although not illustrated in each figure, each of the
three-way stopcocks 51 to 55 may be configured to be switchable by
drive transmitted from a drive source, such as a motor. The
switching of each flow channel may be realized by a valve and the
like other than the three-way stopcocks. The three-way stopcocks 51
and 54 are equivalent to the first switching portions and the
three-way stopcocks 52, 53, and 55 are equivalent to the second
switching portions.
[Culturing of Pluripotent Stem Cells]
[0060] The culture system 10 can be used for culturing pluripotent
stem cells. The pluripotent stem cells which can be cultured by the
culture system 10 are not particularly limited insofar as the
pluripotent stem cells are undifferentiated cells having
"self-replication ability" which allows proliferation while
maintaining the undifferentiated state and
"differentiation/pluripotency" which allows differentiation into
all the three germ layers. Examples of such pluripotent stem cells
include, for example, in addition to ES cells and induced
pluripotent stem cells (iPS cells), mutipotent germline stem (mGS)
cells derived from primordial germ cells, multipotent adult
progenitor cells (MAPC) isolated from the bone marrow, and the
like. The ES cells may be ES cells generated from somatic cells by
nuclear reprogramming. Among the above, the ES cells or the iPS
cells are preferable. The culturing by the culture system 10 can be
applied to arbitrary mammals in which the pluripotent stem cells
are established or can be established. Examples of such mammals
include, for example, human beings, mice, apes, pigs, rats, dogs,
and the like and human beings or mice are preferable and human
beings are particularly preferable.
[0061] The ES cells can be established by taking out an inner cell
mass from the blastocyst of the fertilized ovum of a target animal,
and then culturing the inner cell mass on a fibroblast feeder. The
maintenance of cells by subculturing can be performed using a
culture solution to which substances, such as a leukemia inhibitory
factor (LIF)) and a basic fibroblast growth factor (bFGF), are
added. Methods for establishing and maintaining the ES cells of
human beings and apes are described in, for example, U.S. Pat. No.
5,843,780; Thomson J A, et al. (1995), Proc Natl. Acad. Sci. USA.
92: 7844-7848; Tomson J A, et al. (1998), Science. 282: 1145-1147;
H. Suemori Et Al. (2006), Biochem. Biophys. Res. Commun., 345:
926-932; M. Ueno et al. (2006), Proc. Natl. Acad. Sci. USA, 103:
9554-9559; H. Suemori et al. (2001), Dev. Dyn., 222: 273-279; H.
Kawasaki et al. (2002), Proc. Natl. Acad. Sci. USA, 99: 1580-1585;
Klimanskaya I, et al. (2006), Nature. 444: 481-485, and the
like.
[0062] Using as the culture solution for creating the ES cells, a
DMEM/F-12 culture solution (or a synthetic-medium: mTeSR, Stem Pro,
and the like) supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM
essential amino acids, 2 mM L-glutamic acid, 20% KSR, and 4 ng/mL
bFGF, for example, and the human ES cells can be maintained at
37.degree. C. under a wet atmosphere of 2% CO2 and 98% air (O.
Fumitake et al. (2008), Nat. Biotechnol., 26: 215-224). The ES
cells need to be subcultured every 3 or 4 days. In this case, the
subculturing can be carried out using 0.25% trypsin and 0.1 mg/mL
collagenase IV in PBS containing 1 mM CaCl2 and 20% KSR, for
example.
[0063] The selection of the ES cells can be generally performed
based on the expression of gene markers, such as alkaline
phosphatase, Oct-3/4, and Nanog, as the index by a Real-Time PCR
method. In particular, in the selection of the human ES cells, the
expression of gene markers, such as Oct-3/4, Nanog, and ECAD, can
be used as the index (E. Kroon et al. (2008), Nat. Biotechnol., 26:
443-452).
[0064] As a human ES cell stock, WA01 (H1) and WA09 (H9) are
available from WiCell Research Institute and KhES-1, KhES-2, and
KhES-3 are available from Institute for Frontier Medical Science,
Kyoto University (Kyoto, Japan), for example.
[0065] Sperm stem cells are pluripotent stem cells derived from the
testis and are cells serving as the origin for sperm formation. The
cells can be differentiation-induced into cells of various series
as in the ES cells. For example, the cells have a property which
allows creation of a chimera mouse when transplanted to a mouse
blastocyst (M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:
612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012). The
cells can be self-replicated with a culture solution containing a
glial cell line-derived neurotrophic factor (GDNF) and also the
sperm stem cells can be obtained by repeating subculturing under
the same culture condition as the culture conditions of the ES
cells (Masanori Takebayashi et al. (2008), Jikken Igaku
(Experimental Medicine), Volume 26, No. 5 (extra edition), pp.
41-46, and YODOSHA CO., LTD. (Tokyo, Japan)).
[0066] Embryonic germ cells are established from primordial germ
cells in a fetal period and having the same pluripotency as that of
the ES cells and, can be established by culturing primordial germ
cells in the presence of substances, such as LIF, bFGF, and a stem
cell factor (Y. Matsui et al. (1992), Cell, 70: 841-847; J. L.
Resnick et al. (1992), Nature, 359: 550-551).
[0067] The induced pluripotent stem (iPS) cells are artificial stem
cells derived from somatic cells which can be created by
introducing a specific reprogramming factor into somatic cells in
the form of DNA or protein and which have almost the same
properties as those of the ES cells, e.g.,
differentiation/pluripotency and proliferation potency by
self-replication (K. Takahashi and S. Yamanaka (2006) Cell, 126:
663-676; K. Tkahashi et al. (2007), Cell, 131: 861-872; J. Yu et
al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al. Nat.
Biotechnol. 26: 101-106 (2008; International publication No. WO
2007-069666). The reprogramming factor may be configured from genes
which are specifically expressed in the ES cells, genes which play
an important for maintaining undifferentiation gene products
thereof, non-cording RNA, or the ES cells, gene products thereof,
non-cording RNA, or low molecular weight compounds. Examples of
genes contained in the reprogramming factors include, for example,
Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc,
L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin,
Lin28b, Sall1, Sall14, Esrrb, Nr5a2, Tbx3, or Glis1. These
reprogramming factors may be used alone or in combination of two or
more kinds thereof. Examples of combinations of the reprogramming
factors include combinations described in International Publication
No. WO 2007/069666, International Publication No. WO 2008/118820,
International Publication No. WO 2009/007852, International
Publication No. WO 2009/032194, International Publication No. WO
2009/058413, International Publication No. WO 2009/057831,
International Publication No. WO 2009/075119, International
Publication No. WO 2009/079007, International Publication No. WO
2009/079007, International Publication No. WO 2009/091659,
International Publication No. WO 2009/101084, International
Publication No. WO 2009/101407, International Publication No. WO
2009/102983, International Publication No. WO 2009/114949,
International Publication No. WO 2009/117439, International
Publication No. WO 2009/126250, International Publication No. WO
2009/126251, International Publication No. WO 2009/126655,
International Publication No. WO 2009/157593, International
Publication No. WO 2010/009015, International Publication No. WO
2010/033906, International Publication No. WO 2010/033920,
International Publication No. WO 2010/042800, International
Publication No. WO 2010/050626, International Publication No. WO
2010/056831, International Publication No. WO 2010/056831,
International Publication No. WO 2010/068955, International
Publication No. WO 2010/098419, International Publication No. WO
2010/102267, International Publication No. WO 2010/111409,
International Publication No. WO 2010/111422, International
Publication No. WO 2010/115050, International Publication No. WO
2010/124290, International Publication No. WO 2010/147395,
International Publication No. WO 2010/147612, Huangfu D, et al.
(2008), Nat. Biotechnol., 26: 795-797; Shi Y et al. (2008), Cell
Stem Cell, 2: 525-528; Eminli S et al. (2008), Stem Cells. 26:
2467-2474; Huangfu D et al. (2008), Nat. Biotecnol. 26: 1269-1275;
Shi Y et al. (2008), Cell Stem Cell, 3: 568-574; Zhao Y et al.
(2008), Cell Stem Cell, 3: 475-479; Marson A, (2008), Cell Stem
Cell, 3: 132-135; Feng B et al. (2009), Nat Cell Biol. 11: 197-203;
R. L. Judson et al. (2009), Nat. Biotech., 27: 459-461; Lyssiotis C
A et al. (2009): Proc. Natl. Acad Sci. USA. 106: 8912-8917; Kim J B
et al. (2009), Nature. 461: 643-649; Ichida J K et al. (2009), Cell
Stem Cell. 5: 491-503; Heng J C et al. (2010), Cell Stem Cell. 6:
167-174; Han J et al. (2010), Nature. 463: 1096-1100; Mali P et al.
(2010), Stem Cells. 28: 713-720; Maekawa Met al. (2011), Nature.
474: 225-229.
[0068] The reprogramming factors include factors to be used for the
purpose of increasing the establishment efficiency of histone
deacetylase (HDAC) inhibitors (for example, low molecular weight
inhibitors, such as valproic acid (VPA), tricostatin A, sodium
butyrate, MC1293, and M344, nucleic acidic expression inhibitors,
such as siRNA and shRNA against HDAC (for example, HDAC1 si RNA
Smartpool (Registered Trademark) millipore), HuSH 29 mer shRNA
Constructs against HDAC1 (OriGene and the like), and the like), MEK
inhibitors (for example, PD184352, PD98059, U0126, SL327, and
PD0325901), Glycogen synthase kinase-3 inhibitors (for example,
Bio, CHIR99021), DNA methyl transferase inhibitors (for example,
5-azacytidine), histone methyl transferase inhibitors (for example,
low molecular weight inhibitors, such as BIX-01294, nucleic acidic
expression inhibitors, such as siRNA and shRNA against Suv39HL,
Suv39h2, SetDB1, and G9a, and the like), L-channel calcium agonist
(for example, Bayk8644), butyric acid, TGF.quadrature. inhibitors,
or ALK5 inhibitors (for example, LY364947, SB431542, 616453, and
A-83-01), p53 inhibitors (for example, siRNA and shRNA against
p53), ARID3A inhibitors (for example, siRNA and shRNA against
ARID3A), miRNA, such as miR-291-3p, miR-294, miR-295, and miR-302),
Wnt Signaling (for example, soluble Wnt3a), neuropeptide Y,
prostaglandins (for example, prostaglandin E2 and prostaglandin
J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, DMRTB1, and the like.
In this specification, the factors used for the purpose of
improving the establishment efficiency thereof are not also
particularly distinguished from the reprogramming factors.
[0069] In the case of the form of protein, the reprogramming
factors may be introduced into somatic cells by techniques, such as
lipofection, fusion with cell membrane permeable peptide (for
example, TAT derived from HIV and polyarginine), and
microinjection, for example.
[0070] On the other hand, in the case of the form of DNA, the
reprogramming factors can be introduced into somatic cells using
vectors, such as virus, plasmid, and artificial chromosome, or
techniques, such as lipofection, liposome, and microinjection, for
example. Examples of the virus vectors include retroviras vectors,
lentivirus vectors (Cell, 126, pp. 663-676, 2006; Cell, 131, pp.
861-872, 2007; Science, 318, pp. 1917-1920, 2007), adenovirus
vectors (Science, 322, 945-949, 2008), adeno-associated virus
vectors, sendai virus vectors (International Publication No. WO
2010/008054), and the like. Examples of the artificial chromosome
vectors include human artificial chromosome (HAC), yeast artificial
chromosome (YAC), bacteria artificial chromosome (BAC, PAC), and
the like, for example. As the plasmid, plasmid for mammal cells can
be used (Science, 322: 949-953, 2008). The vectors can contain, in
such a manner that the nuclear reprogramming substances can be
expressed, control sequences of a promoter, an enhancer, a ribosome
junction sequence, a terminator, a polyadenylation site, and the
like and further, selective marker sequences of a drug resistant
gene (for example, a kanamycin resistant gene, an ampicillin
resistant gene, and a puromycin resistant gene), a thymidine kinase
gene, a diphtheria toxin gene, and the like, reporter gene
sequences of green fluorescent protein (GFP), .beta.glucuronidase
(GUS), and FLAG, and the like, etc., as necessary. In order to
break both the genes coding the reprogramming factor or the
promoter and the gene coding the reprogramming factors bonded
thereto after introducing into somatic cells, the vectors may have
a LoxP sequence before and after the sequences.
[0071] In the case of the form of RNA, the reprogramming factors
may be introduced into somatic cells by techniques, such as
lipofection and microinjection, for example, and, in order to
suppress decomposition, RNA into which 5-methyl cytidine and
pseudouridine (TriLink Biotechnologies) are introduced may be used
(Warren L, (2010), Cell Stem Cell. 7: 618-630).
[0072] Culture solutions for iPS cell induction (These culture
solutions can further contain LIF, penicillin/streptomycin,
puromycin, L-glutamine, non-essential amino acids,
.beta.-mercaptoethanol, and the like as appropriate.), commercially
available culture solutions (for example, a culture solution for
mouse ES cell culture (TX-WES culture solution, Thrombo X), a
culture solution for primate ES cell culture (primate ES/iPS cell
culture solution, ReproCELL Incorporated), a serumless culture
medium (mTeSR, Stemcells Technology), and the like.
[0073] As an example of a culturing method, somatic cells and the
reprogramming factor are brought into contact with each other on a
10% FBS containing DMEM or a DMEM/F12 culture solution at
37.degree. C. in the presence of 5% CO.sub.2, the cells are
cultured for about 4 to 7 days, the cells are seeded again on
feeder cells (for example, mitomycin C treated STO cells, SNL
cells, and the like), and then, about 10 days after the contact
between the somatic cells and the reprogramming factor, the cells
are cultured in a culture solution for culturing bFGF containing
primate ES cells, so that an iPS-like colony can be produced about
30 to 45 days or longer after the contact, for example.
[0074] Or, the cells are cultured on feeder cells (for example,
mitomycin C treated STO cells, SNL cells, and the like) in a 10%
FBS containing DMEM culture solution (The culture solution can
further contain LIF, penicillin/streptomycin, puromycin,
L-glutamine, non-essential amino acids, .beta.-mercaptoethanol, and
the like as appropriate.) at 37.degree. C. in the presence of 5%
CO.sub.2, so that an ES-like colony can be produced about 25 to 30
days or longer later. Preferably, a method using somatic cells
themselves to be reprogrammed in place of the feeder cells
(Takahashi K et al. (2009), PLoS One. 4: e8067 or International
Publication No. WO 2010/137746) or a method using an extracellular
matrix (for example, Laminin (International Publication No. WO
2009/123349), Matrigel (BD)) in place of the feeder cells are
mentioned.
[0075] In addition thereto, a method for performing culturing using
a culture medium not containing serum is also mentioned (Sun N et
al. (2009), Proc Natl Acad Sci USA. 106: 15720-15725). Furthermore,
in order to increase the establishment efficiency, iPS cells may be
established under low oxygen conditions (oxygen concentration of
0.1% or more and 15% or less) (Yoshida Y et al. (2009), Cell Stem
Cell. 5: 237-241, International Publication No. WO
2010/013845).
[0076] During the culturing, the culture solution is exchanged with
a fresh culture solution once every day from the second day after
starting the culturing. The number of cells of the somatic cells to
be used for nuclear reprogramming is not limited and is in the
range of about 5.times.10.sup.3 to 5.times.10.sup.6 cells per 100
cm.sup.2 of a culture dish.
[0077] The iPS cells can be selected in accordance with the shape
of the formed colony. On the other hand, when a drug resistant gene
expressed in connection with a gene (for example, Oct3/4, Nanog)
expressed when somatic cells are reprogrammed is introduced as a
marker gene, the established iPS cells can be selected by
performing culturing in a culture solution (selective culture
solution) containing a corresponding drug. When the marker gene is
a fluorescent protein gene, the established iPS cells can be
selected by observation under a fluorescence microscope. In the
case of a luciferase gene, the established iPS cells can be
selected by adding a luminescent substrate. In the case of a
chromogenic enzyme gene, the established iPS cells can be selected
by adding a chromogenic substrate.
[Suspension Culture of Pluripotent Stem Cells (Culturing
Process)]
[0078] The pluripotent stem cells prepared as described above are
suspended and cultured by the culture system 10. Specifically, the
culture bag 11 is charged with the pluripotent stem cells together
with a culture medium, and then suspended and cultured until the
average diameter of cell masses of the pluripotent stem cells
reaches about 200 to 300 .mu.m. The term "about" used herein means
that .+-.10% is permitted. When the diameter of the cell mass
exceeds 300 .mu.m, there are problems that a microenvironment is
formed due to cytokine and the like secreted by the cells, and
differentiation is induced and further necrosis occurs in the
central portion of the cell mass, so that the recovery rate of raw
cells decreases. On the other hand, when the lower limit of the
average diameter of the cell masses is not particularly limited
insofar as the average diameter is larger than the average diameter
of the cell masses when starting the suspension and culturing (when
suspending in the case of suspending and culturing after
subculturing). In consideration of the yield of the pluripotent
stem cells, it is preferable to continue the culturing until the
average diameter reaches about 200 .mu.m or more.
[0079] As the culture medium for suspension culture, a culture
medium having the same composition as that of the culture medium
for adhesion culture described above can be used. Preferably, in
order to prevent movement of the cell masses and adhesion of the
cell masses, it is desirable to give moderate viscosity to the
culture medium. Herein, the "moderate viscosity" means the
viscosity at which the culture medium exchange is not hindered and
the adhesion of the cell masses do not occur.
[0080] A means for giving viscosity to the culture medium is not
particularly limited. The viscosity can be given to the culture
medium by adding a water soluble polymer to the culture medium with
a preferable concentration, for example. As the water soluble
polymer, any water soluble polymer can be used insofar as moderate
viscosity can be given to the culture medium and the cells are not
adversely affected (no cytotoxicity) in a concentration range where
viscosity can be given. Examples of the water soluble polymer
include, for example, polysaccharides, such as cellulose and
agarose, ethers of polysaccharides, such as methyl cellulose, ethyl
cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,
hydroxyethyl methyl cellulose, hydroxypropyl methylcellulose,
hydroxyethyl ethyl cellulose, hydroxypropylethyl cellulose, ethyl
hydroxyethyl cellulose, dihydroxypropyl cellulose, and hydroxyethyl
hydroxypropyl cellulose, synthetic polymers, such as
polyacrylamide, polyethylene oxide, polyvinyl pyrrolidone, an
ethylene glycol/propylene glycol copolymer, polyethylene imine
polyvinyl methyl ether, polyvinyl alcohol, polyacrylic acid, and a
maleic acid copolymer, biopolymers, such as collagen, gelatin,
hyaluronic acid, dextran, alginic acid, carrageenan, and starch, or
artificial polymers (for example, elastin-like peptide and the
like) imitating them. These water solubility polymers may be used
alone and can also be used as a mixture of several kinds of the
water soluble polymers. Moreover, copolymers of these water
solubility polymers may be used. Methyl cellulose, polyethylene
glycol, polyvinyl pyrrolidone, carboxymethylcellulose, or a mixture
thereof is preferable and methyl cellulose is more preferable.
[0081] For example, when methyl cellulose is added to the culture
medium in order to give viscosity thereto, the concentration of the
methyl cellulose is preferably higher than 0.25 w/v % and lower
than 0.5 w/v %. When the concentration of methyl cellulose is 0.25
w/v % or less, the viscosity is excessively low, and thus a desired
effect cannot be obtained. When the concentration is 0.5 w/v % or
higher, the culture medium becomes difficult to pass through the
trap portion 14 and the subculturing filter portion 15. The
concentration of the methyl cellulose is preferably 0.26 to 0.3 w/v
% and particularly preferably 0.28 w/v %. Even when using another
water soluble polymer, a person skilled in the art can select
another water soluble polymer and a concentration in order to
obtain the moderate viscosity described above.
[0082] In another preferable aspect, a temperature-rise type
temperature sensitivity hydro-gel can be used as the water soluble
polymer. The "temperature-rise type temperature sensitivity
hydro-gel" refers to a hydro-gel which is liquid at a low
temperature and which exhibits a reversible sol-gel phase
transition in which the hydro-gel is gelated by raising the
temperature and is solated again by cooling the gel to room
temperature. Examples of the temperature-rise type temperature
sensitivity hydro-gel include "Mebiol gel" series (Trade name,
Mebiol Inc.) which exhibit a gelling transition temperature of 27
to 32.degree. C. and the like but the temperature-rise type
temperature sensitivity hydro-gel is not limited thereto. When
using the temperature-rise type temperature sensitivity hydro-gel,
the hydro-gel is added at a concentration at which sufficient
viscosity for preventing movement of floating cell masses and
adhesion of cell masses can be given, the cells are proliferated to
a size suitable for subculturing by suspension culture, the culture
solution is solated by cooling the same to a temperature equal to
or lower than the gelling transition temperature, and then the
cells are made to pass through the trap portion 14 or the
subculturing filter portion 15, whereby the cells can be easily
recovered.
[0083] The culturing is performed by dissociating the pluripotent
stem cells subjected to the adhesion culture by enzyme treatment,
seeding the cells into the culture bag 11 in such a manner that the
cell density is about 0.5 to 50+104 cells/cm2 and preferably about
1 to 10+104 cells/cm2, and then culturing the cells in a CO.sub.2
environment in the culture system 10 under a CO.sub.2 atmosphere
having a concentration of about 1 to 10% and preferably about 2 to
5% at about 30 to 40.degree. C. preferably about 37.degree. C. for
1 to 7 days, preferably 3 to 6 days, and more preferably 4 or 5
days.
[0084] During the culturing described above, it is preferable to
exchange the culture medium in the culture bag 11 with a fresh
culture medium every 1 or 2 days. When exchanging the culture
medium, the culture medium is made to flow out of the culture bag
11 into the waste liquid container 12 (arrow 101). Due to the fact
that the three-way stopcocks 51 to 54 are operated, the flow
channel from the culture bag 11 to the waste liquid container 12
through the trap portion 14 is opened. Due to the fact that the
pump 16 is operated, the culture medium flows into the waste liquid
container 12 through the first flow channel 41 and the second flow
channel 42 from the culture bag 11 at a fixed flow rate. The flow
rate is preferably a fixed flow rate in the range of about 5 to 50
mL per minute and more preferably a fixed flow rate in the range of
about 8 to 10 mL per minute.
[0085] The cell masses of the pluripotent stem cells flow out of
the culture bag 11 together with the culture medium. However, since
the cell masses are trapped by the trap portion 14, the cell masses
do not flow into the waste liquid container 12. Specifically, the
cell masses of the pluripotent stem cells flow into the internal
space of the body 20 together with the culture medium from the
inflow port 21 in the lower side of the body 20. In the internal
space of the body 20, the cell masses stay in the lower side of the
internal space of the body 20 by own weight. On the other hand, the
culture medium flows out of the outlet port 22 from the internal
space of the body 20. The cell masses which reach the upper side in
the internal space of the body 20 are blocked by the mesh 23, and
do not flow out of the outlet port 22.
[0086] When all the culture media in the culture bag 11 are made to
flow out, the three-way stopcock 51 is switched, so that the
culture bag 11 and the fresh culture medium container 13 are
connected to each other through the trap portion 14. Due to the
fact that the pump 16 is operated, a fresh culture medium flows
from the fresh culture medium container 13 into the culture bag 11
through the first flow channel 41, the second flow channel 42, and
the third flow channel 43 at a fixed flow rate (arrow 102). The
flow rate is preferably a fixed flow rate in the range of about 50
to 150 mL per minute and more preferably a fixed flow rate in the
range of about 80 to 100 mL per minute.
[0087] The fresh culture medium which flows out of the fresh
culture medium container 13 flows into the internal space of the
body 20 from the outlet port 22 of the trap portion 14. Then, the
fresh culture medium flows out of the inflow port 21, and then
flows into the culture bag 11 through the first flow channel 41
together with the cell masses of the pluripotent stem cells staying
in the lower side of the internal space of the body 20 (arrow 103).
Thus, the cell masses of the pluripotent stem cells trapped by the
trap portion 14 flow into the culture bag 11 together with the
fresh culture medium.
[0088] When the cell masses clog the mesh 23 of the trap portion
14, a backwashing culture medium may be made to flow into the trap
portion 14 from the backwashing container 17 in order to remove the
clogging cell masses.
[0089] The backwashing culture medium is a culture medium to be
used for the suspension culture described above and is preferably a
culture medium to which the water soluble polymer is not added, for
example. Due to the backwashing culture medium having low viscosity
passes through the mesh 23 of the trap portion 14, the cell masses
clogging the mesh 23 are easily separated from the mesh 23. The
flow amount and the flow rate of the backwashing culture medium are
selected as appropriate according to the clogging level of the mesh
23.
[0090] Specifically, when the culture medium is made to flow out of
the culture bag 11 into the waste liquid container 12 through the
trap portion 14, the operation of the pump 16 is suspended, and
then the three-way stopcock 54 is switched, so that the backwashing
container 17 and the pump 16 are connected to each other through
the sixth flow channel 46. Then, due to the fact that the pump 16
is operated again, the backwashing culture medium from the
backwashing container 17 flows through the second flow channel 42
toward the fresh culture medium container 13 through the sixth flow
channel 46 (arrow 104). When only a predetermined amount of the
backwashing culture medium flows into the second flow channel 42,
the operation of the pump 16 is suspended, and then the three-way
stopcock 54 is switched, so that the culture bag 11 and the fresh
culture medium container 13 are connected to each other through the
trap portion 14. Then, due to the fact that the pump 16 is operated
in the opposite direction (direction indicated by the arrow 102),
specifically in a direction from the fresh culture medium container
13 toward the trap portion 14, the backwashing culture medium
flowing into the second flow channel 42 flows into the internal
space of the body 20 through the outlet port 22 of the trap portion
14. At this time, the cell masses clogging the mesh 23 of the trap
portion 14 are separated due to the backwashing culture medium.
Then, the direction of the pump 16 is switched, so that the culture
medium is made to flow out of the culture bag 11 to the waste
liquid container 12 side in the same manner as described above.
[0091] The culture medium in the culture bag 11 is exchanged with a
fresh culture medium every 1 or 2 days in the suspension
culture.
[0092] For example, it is supposed that the average diameter of the
cell masses of the pluripotent stem cells when starting the
suspension culture is about 80 .mu.m, and the cells are
proliferated until the average diameter reaches about 250 .mu.m, it
is necessary to amplify the number of cells in the cell masses by
about 3.sup.3=27 times. For example, human ES cells are divided
once in about 24 hours. Therefore, it is considered that, when the
cells are cultured for 4 or 5 days, the cells are proliferated to a
desired size also in terms of calculation.
[Subculturing of Pluripotent Stem Cells]
[0093] The cell masses of pluripotent stem cells having a uniform
size in which the average diameter of the cell masses of the
pluripotent stem cells is about 200 to 300 .mu.m are then divided
into small cell masses having an almost uniform size in which the
average diameter is about 80 to 120 .mu.m to be subcultured. The
term "about" used herein means that .+-.20% is permitted. When the
cell masses are divided in such a manner that the average diameter
of the cell masses is 50 .mu.m or less, the cells are likely to
cause cell death, such as apoptosis, and thus the average diameter
is not preferable. The upper limit of the average diameter after
division is not particularly limited. However, when the size is
larger, the amplification efficiency by the suspension culture
after the following subculturing further decreases. Therefore, the
average diameter is preferably about 120 .mu.m or less and
particularly preferably about 40 to 80 .mu.m.
[0094] Due to the fact that the three-way stopcocks 51 to 54 are
operated after the cells are proliferated until the average
diameter of the cell masses of the pluripotent stem cells is
amplified to about 200 to 300 .mu.m in the culture bag 11, the flow
channel from the culture bag 11 to the waste liquid container 12
through the trap portion 14 is opened. Due to the fact that the
pump 16 is operated, the culture medium flows into the waste liquid
container 12 through the first flow channel 41 and the second flow
channel 42 from the culture bag 11 at a fixed flow rate (arrow
101). The flow rate is preferably a fixed flow rate in the range of
about 5 to 50 mL per minute and more preferably a fixed flow rate
in the range of about 8 to 10 mL per minute.
[0095] The cell masses of the pluripotent stem cells flow out of
the culture bag 11 together with the culture medium. However, since
the cell masses are trapped by the trap portion 14 as described
above, the cell masses do not flow into the waste liquid container
12. When all the culture media in the culture bag 11 are made to
flow out, the three-way stopcocks 51, 52, and 53 are switched, so
that the culture bag 11 and the fresh culture medium container 13
are connected to each other through the trap portion 14 and the
subculturing filter portion 15.
[0096] Due to the fact that the pump 16 is operated, a fresh
culture medium flows toward the culture bag 11 through the second
flow channel 42, the third flow channel 43, the fourth flow channel
44, and the fifth flow channel 45 from the fresh culture medium
container 13 at a fixed flow rate (arrow 102). The fresh culture
medium which flows out of the fresh culture medium container 13
flows into the internal space of the body 20 from the outlet port
22 of the trap portion 14, and then flows out of the inflow port 21
together with the cell masses of the pluripotent stem cells staying
in the lower side of the internal space of the body 20 in the same
manner as described above. Thus, the cell masses of the pluripotent
stem cells trapped by the trap portion 14 flow into the
subculturing filter portion 15 with the fresh culture medium (arrow
105).
[0097] The cell masses of the pluripotent stem cells which reach
the subculturing filter portion 15 together with the fresh culture
medium from the trap portion 14 are divided when passing through
the mesh 31. The flow rate when letting the cell masses of the
pluripotent stem cells pass through the mesh 31 is preferably a
fixed flow rate in the range of about 90 to 300 mL/min. The term
"about" used herein means that .+-.10% is permitted. The flow rate
is the flow rate in the second flow channel 42 or the fourth flow
channel 44 caused by the driving force of the pump 16. When the
flow rate is lower than about 90 mL/min, the number of the cell
masses which cannot pass through the mesh 31 increases, so that the
recovery rate of the pluripotent stem cells collected by the
culture bag 11 decreases. The upper limit of the flow rate is not
particularly limited. However, when the flow rate is higher than
about 300 mL/min, the average diameter of the cell masses passing
through the mesh 31 tends to be small.
[0098] When the area (effective area) through which the cell masses
of the pluripotent stem cells can pass in the mesh 31 is enlarged,
the cross-sectional area of the space where the mesh 31 is present
in the body 30 to the cross-sectional area of the fourth flow
channel 44 becomes large, and, as a result, the flow rate of the
cell masses (culture medium) which pass through the region tends to
be lower as approaching the periphery of the mesh 31. Accordingly,
the area of the cell mass passing through the mesh 31 may also
change when the effective area of the mesh 31 changes. Therefore,
the flow rate when letting the cell masses of the pluripotent stem
cells pass through the mesh 31 may be grasped as a flow rate per
unit square cm of the mesh 31. For example, when the effective
diameter of the mesh 31 having a disk shape is 11 mm, the flow rate
per unit square cm of the mesh 31 is preferably in the range of
about 95 to 315 mL/min.
[0099] The process of letting the cell mass of the pluripotent stem
cells pass through the mesh 31 with a fresh culture medium to
divide the cell mass is equivalent to the first dividing
process.
[0100] When the cell masses clog the mesh 31 of the subculturing
filter portion 15, a backwashing culture medium may be made to flow
into the subculturing filter portion 15 from the backwashing
container 18 in order to remove the clogging cell masses.
Specifically, the operation of the pump 16 is suspended, and then
the three-way stopcock 55 are switched, so that the backwashing
container 18 and the subculturing filter portion 15 are connected
to each other through the seventh flow channel 47. Then, when the
pump 16 is operated in the opposite direction (rightward in the
subculturing filter portion 15 of FIG. 1), the backwashing culture
medium from the backwashing container 18 back-flows through the
mesh 31 toward the inflow port 32 from the outlet port 33 of the
subculturing filter portion 15 (arrow 106).
[0101] The backwashing culture medium is a culture medium to be
used for the suspension culture described above and is preferably
one to which the water soluble polymer is not added, for example.
Due to the fact that the backwashing culture medium having low
viscosity passes through the mesh 31 of the subculturing filter
portion 15, the cell masses clogging the mesh 31 are easily
separated from the mesh 31. The flow amount and the flow rate of
the backwashing culture medium are selected as appropriate
according to the clogging level of the mesh 31.
[0102] After the backwashing is completed, the operation of the
pump 16 is suspended, and then the three-way stopcock 55 is
switched, so that the culture bag 11 and the subculturing filter
portion 15 are connected to each other. Then, due to the fact that
the pump 16 is operated in a forward direction (leftward in the
subculturing filter portion 15 of FIG. 1) again (arrow 105), a
fresh culture medium is made to flow out of the fresh culture
medium container 13 into the culture bag 11 in the same manner as
described above.
[0103] The process of removing the cell masses of the pluripotent
stem cells once from the mesh 31 together with the backwashing
culture medium, and then letting the cell masses pass through the
mesh 31 again to divide the same as described above is equivalent
to the second dividing process.
[0104] The cell masses of the pluripotent stem cells which are
divided into a size suitable for subculturing flow into the culture
bag 11 together with the fresh culture medium. Thus, the
pluripotent stem cells are subcultured.
Working Effects of this Embodiment
[0105] According to the culture system 10 of pluripotent stem cells
according to this embodiment, a large amount of pluripotent stem
cells can be cultured and an automated system can be realized.
[0106] Moreover, even when cell masses of pluripotent stem cells
which reach the subculturing filter portion 15 from the trap
portion 14 together with a fresh culture medium in subculturing
clog the mesh 31 without being divided, a backwashing culture
medium is made to flow into the mesh 31 from the backwashing
container 18, and then the clogging cell masses are carried by the
backwashing culture medium to be separated from the mesh 31. Thus,
the clogging of the mesh 31 of the subculturing filter portion 15
can be eliminated.
[0107] Moreover, by letting a culture medium flow into the
subculturing filter portion 15 at a fixed flow rate of 90 mL or
more per minute in subculturing, the recovery rate of pluripotent
stem cells improves.
EXAMPLES
[0108] [Flow Rate of Culture Medium Passing through Subculturing
Filter Portion 15]
[0109] The cell recovery rate when the flow rate of a culture
medium passing through the subculturing filter portion 15 was set
to each of 10 mL, 50 mL, 90 mL, 150 mL, and 300 mL per minute was
measured. Since the effective diameter of the mesh 31 was 11 mm,
the flow rate was 10.5 mL, 52.6 mL, 94.7 mL, 157.9 mL, and 315.8 mL
per minute when converted to the flow rate per unit square cm of
the mesh 31.
(Preparation of Reagent)
[0110] 100 mL of a supplement (VERITAS Corporation, ST-05850) was
added to 350 mL of a base culture medium (VERITAS Corporation,
ST-05850), and then sufficiently stirred. Furthermore, 500 mL of a
Y-27632 solution (SIGMA, Y0503-5MG) was added in such a manner that
final concentration was 10 .mu.m. Furthermore, 50 mL of methyl
cellulose liquid (R&D, HSC001) was added, and then sufficiently
stirred to prepare a subculturing culture medium.
[0111] 50 mL of methyl cellulose liquid was added to 450 mL of a
DMEM/F12 culture medium (Life Technologies), and then sufficiently
stirred to obtain a backwashing culture medium.
(Subculture Operation)
[0112] The culture medium was made to flow out of the culture bag
11 into the trap portion 14 together with cell masses of
pluripotent stem cells. The cell masses were trapped in the trap
portion 14, and then only the culture medium was made to flow to
the waste liquid container 12 side. In this operation, the culture
medium was made to flow out of the waste liquid container 12 while
letting a backwashing culture medium back-flow into the trap
portion 14 from the backwashing container 17 as appropriate to
eliminate clogging of the mesh 23. Then, a fresh culture medium was
made to flow out of the fresh culture medium container 13 into the
culture bag 11 through the trap portion 14, and then the
subculturing filter portion 15. Thus, the cell masses trapped in
the trap portion 14 were made to pass through the mesh 31 (formed
with stainless steel, Pore size of 50 .mu.m, Opening ratio of 64%)
of the subculturing filter portion 15 to divide the cell masses to
be subcultured. By letting the backwashing culture medium back-flow
into the subculturing filter portion 15 from the backwashing
container 18 as appropriate to eliminate the clogging of the mesh
31, and then letting the same pass through the mesh 31 again in
subculturing, the clogging cell masses were also divided.
(Determination of Recovery Rate by Calculation)
[0113] The recovery rate of the pluripotent stem cells collected by
the culture bag 11 after subculturing was determined by
calculation. The recovery rate was determined by the following
equation.
(Recovery rate)=(Total number of cells after subculturing)/(Total
number of cells before subculturing)
[0114] The results are shown in FIG. 4. As is clear from FIG. 4,
the tendency was confirmed that the recovery rate further improved
as the flow rate when the culture medium passed through the
subculturing filter portion 15 became higher and the recovery rate
at flow rates of 90 mL or more per minute was fixed.
(Size of Cell Mass after Subculturing)
[0115] The size and the distribution of the cell masses of the
pluripotent stem cells collected by the culture bag 11 after
subculturing were measured. The results are shown in FIG. 5. As is
clear from FIG. 5, when the flow rate was low, the distribution of
the cell masses having a size larger than the pore size of the mesh
31 tended to increase, and a distribution of the cell mass sizes
tended to spread as a whole. When the flow rate was high, a
distribution of the cell masses having a size close to the pore
size of the mesh 31 tended to increase, and a distribution of the
cell mass sizes tended to narrow as a whole.
[Opening Ratio of Mesh 31 of Subculturing Filter Portion 15]
[0116] For the mesh 31 of the subculturing filter portion 15, those
having an opening ratio of 72% (formed with stainless steel, Pore
size of 46 .mu.m), 71% (formed with stainless steel, Pore size of
55 .mu.m), 64% (formed with stainless steel, Pore size of 50
.mu.m), and 58% (formed with PET, Pore size of 98 .mu.m) were used,
and the subculturing operation described above was performed about
each mesh. The flow rate of the culture medium passing through the
subculturing filter portion 15 was set to 90 mL per minute.
[0117] The results of determining the recovery rate for the mesh 31
of each opening ratio by calculation are shown in FIG. 6. As is
clear from FIG. 6, the tendency was confirmed that, when the
material of the mesh 31 was the same (formed with stainless steel),
the recovery rate further improved when the opening ratio was
higher.
REFERENCE SIGNS LIST
[0118] 10 Culture system
[0119] 11 Culture bag
[0120] 12 Waste liquid container
[0121] 13 Fresh culture medium container
[0122] 14 Trap portion
[0123] 15 Subculturing filter portion
[0124] 16 Pump
[0125] 17,18 Backwashing container
[0126] 31 Mesh
* * * * *